Cell migration is a very influential process during embryonic development as it results in rearrangement of cells from one area of the embryo to another, effectively controlling cell-cell interactions to drive cell differentiation and organogenesis. The shape of most complex organ systems arises from the directed migration of cohesive cell groups. Therefore, cell migration must be regulated temporally and spatially for organisms to develop properly. The overlying goal of our research objective is to provide insight into how cells within a migrating group sense their environment and how this contributes to their collective movement. To accomplish this goal, we must first develop methodology to facilitate study of caudal visceral mesoderm (CVM) cell migration in Drosophila. We contend that CVM cell migration serves as an excellent model system to study how cell migration shapes gut formation. CVM cells exhibit directed cell migration from the posterior-most position of the embryo toward the anterior as two distinct groups on either side of the body. These cells undergo the longest migration in all of Drosophila embryogenesis, but little is understood about how they are directed along their course. Their migration is necessary to position cells along the entire length of the gut, as these cells form longitudinal muscles that must completely ensheath the gut. Our goal is to define methodology that will enable study of the CVM cell migration, to develop it as a system for studying how cell migration influences organogenesis. Our first aim will be to define methods that support live in vivo imaging of CVM cell nuclei to provide cell tracking data, as well as imaging of CVM cell membranes to provide insight into how cells interact with their environment. In the second aim, we will define quantitative methods to analyze cell movements and cellular projections to provide a quantitative description of cells' migration. Cell tracking data and cell protrusion number and orientation can provide important information about the cell migration process in wildtype embryos, and can be used as a reference to interpret mutant phenotype. In addition, Drosophila presents an excellent system to assay loss of function phenotypes for genes with respect to a particular biological process. Therefore, in our third aim, we will optimize an RNAi strategy utilizing short hairpin RNA transgenes and tissue- specific Gal4 constructs which, when present together in the genome, support RNAi to effectively reduce expression of genes of interest within the CVM. In our last aim, we present a novel approach for creating mutant clones within developing Drosophila embryos using a light-activated recombinase. This method will allow us to assay gene function both spatially and temporally within the migrating cell collective. Such methodology would be powerful; generally applicable toward the study of cell migration and useful to the developmental biology community at large. Development of the gastrointestinal tract of animals is highly similar, so studies of how CVM cell migration influences gut formation in Drosophila have the potential to have far-reaching insight into human development. PUBLIC HEALTH RELEVANCE: Uncontrolled cell migration can lead to congenital defects in vertebrates affecting the heart, vasculature, and gastrointestinal tract. It is possible that congenital defects identified in muscles of the stomach, causing neonatal spontaneous gastric perforation which proceeds rapidly and has a high mortality rate, result from aberrant cell migration earlier in development. As development of the gastrointestinal tract of animals is highly similar, studies of how CVM cell migration influences gut formation in Drosophila have the potential for far- reaching implications.